Enveloped virus vaccine and method for production

ABSTRACT

The present invention provides methods of production of a purified enveloped virus antigen. In particular, it provides purified Ross River Virus (RRV) antigens, and vaccines comprising purified, inactivated Ross River Virus (RRV) antigen.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/657,848, filed Jan. 24, 2007 which is a division of U.S. patentapplication Ser. No. 10/006,671, filed on Dec. 10, 2001, now U.S. Pat.No. 7,195,905, all of the above are incorporated by reference in theirentirety.

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REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

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FIELD OF THE INVENTION

The present invention is directed to methods of production of a purifiedenveloped virus antigen, for example, Ross River Virus (RRV) antigen,and to a vaccine comprising purified, inactivated Ross River Virus (RRV)antigen, wherein the RRV antigen is free of heterologous nucleic acidand protein contaminants from the cells and the cell culture.

BACKGROUND OF THE INVENTION

Safe vaccine production, particular for human administration requiresthe growth of large quantities of virus produced in high yields from ahost system, as well as efficient purification and inactivation methods.Serum or serum-derived substances such as albumin, transferrin orinsulin and/or proteins from animal or human sources added to a cellculture medium may contain undesirable agents that can contaminate theculture and the biological material produced therefrom. One of the majorconcerns for the production of biologicals, such as vaccines orrecombinant products, is the potential risk of contaminants such asBovine Spongiform Encephalopathy (BSE). Furthermore, the World HealthOrganization (WHO) encourages the use of agents having an inactivatingeffect on the biological activity of DNA. On the basis of a study on thepotential risks associated with biologicals produced in animal cells,the WHO Study Group concluded that levels at up to 10 ng nucleic acidper purified dose can be considered acceptable (1998, WHO, TechnicalReport Series No. 878), but levels of less than 100 pg per dose would bepreferred. However, most viruses used for vaccine production have notbeen purified to preserve their sensitive biological activity which iscritical for efficacy of the vaccine, because efficient purificationmethods seem to influence the immunogenicity and antigenicity of thevirus. In addition, purification methods currently available for virusor virus antigen do not always remove the cell culture contaminantsefficiently. Thus, for each particular virus, an appropriate method hasto be developed and adapted to large scale.

Ross River Virus (RRV) is a mosquito borne Alphavirus which causes adisease in humans known as epidemic polyarthritis (EPA). It is endemicin Australia, with more than 5000 cases presenting each year. Currently,there is no existing vaccine, and mosquito control programs have not hadany noticeable effect in reducing the incidence of disease.

An experimental candidate RRV vaccine previously developed by Yu et al.(1994, Vaccine 12: 1118-1124) and Aaskov et al. (1997, Vaccine 15:1396-1404) has been demonstrated to protect mice from challenge withlive virus. This vaccine is obtained from the supernatant of infectedVERO cells cultivated on a small scale basis in Roller bottles inserum-containing medium. The virus-containing supernatant is centrifugedto remove cellular debris and the supernatant is incubated for virusinactivation with Binary Ethylenimine (BEI) under alkali conditions atpH 8.5-9.0. The inactivated virus suspension is subjected to sucrosegradient centrifugation, the band of virus of the gradient interfacerecovered is resuspended in saline buffer and used for immunizationstudies in mice. However, in the preparation solely subjected tosucrose-gradient purification without any other treatment, cellularcontaminants, such as cellular DNA or proteins, can be still present inthe virus preparation.

The candidate BEI-inactivated Ross River Vaccine described, supra, istested at different concentrations of an adjuvant and without adjuvantin mice. Greater protection is found with the vaccine at higher dosewithout adjuvant than those with an adjuvant. Even at lower dose thenon-adjuvanted vaccine induced significantly higher antibody titerscompared to the adjuvanted. Comparative studies on neutralizing antibodytiters after a second injection of mice with low antigen concentrationsof 0.2 or 2.0 μg of vaccine with and without adjuvant caused researchersto conclude that the adjuvant may suppress at low concentrations theprotective component of a secondary immune response to theBEI-inactivated vaccine.

Because of the need for a safe and effective Ross River Virus vaccinefor human administration, large-scale production of virus and efficientpurification and inactivation methods are required. The presentinvention is directed to the large-scale production of Ross River Virussuitable for preparation of a vaccine for administration to humans.

BRIEF SUMMARY OF THE INVENTION

It is thus an object of the present invention to provide a method forthe production of purified Ross River Virus antigen.

It is also an object of the present invention to provide a method ofproduction of a vaccine comprising purified Ross River Virus beingsuitable for human administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: western blot analysis of efficacy of filtration of Ross RiverVirus harvest on removal of cellular proteins derived from cell culture.FIG. 1A: detection of VERO cell protein with anti-VERO proteinantibodies and FIG. 1B: detection of Ross River Virus antigen withanti-RRV antibodies. It is shown in lane 1: Vero cell lysate as control,lane 2: RRV harvest after centrifugation, lane 3: after filtration, lane4: after BENZONASE endonuclease treatment, lane 5: after sucrosegradient purification.

DETAILED DESCRIPTION OF THE INVENTION

An object of the invention is to provide a purified RRV antigensubstantially free of contaminants derived from the cell culture mediumand the cells of the cell culture, such as cellular proteins andcellular nucleic acid, wherein the purified antigen is used in a vaccineparticularly suitable for human clinical use in a host protectiveamount.

The term “cellular nucleic acid” means a heterogeneous DNA or RNAderived from the cells that have been infected with the virus topropagate the virus.

By “purified Ross River Virus antigen” is meant greater than about 97%purity as determined by SDS-PAGE and western blot analysis withanti-cellular protein specific antibodies and quantification of residualcellular nucleic acid.

The term “substantially free” means that the amount of contaminatingproteins derived from the cells or the cell culture or contaminatingcellular nucleic said amount being below the detection limit of thestate of the art detection method. Western blot analysis anddensitometric determination are used to test the amount of contaminatingcellular proteins. A highly sensitive PCR method as described in U.S.Pat. No. 5,858,658 for nucleic acid quantification, particular forgenomic VERO cell DNA, is used to quantify residual cellular nucleicacid in preparation.

The term “suitable for human clinical use” means that the endotoxincontent for 10 μg antigen is less than about 2 IU, as determined thechromogenic LAL test. In addition, the level of DNA/μg protein antigenin the vaccine dose is less than about 50 pg, preferably less than about20 pg, more preferably less than about 10 pg/μg antigen. Furthermore,the level of cellular contaminants per dose of virus antigen is lessthan about 0.1% of the total protein content, preferably less than about0.05%, more preferably below the detection limit of highly sensitiveanalysis methods such as western blot analysis with specific antibodiesor HPLC analysis.

The “host protective amount” means the critical protective dose of viralantigen in the vaccine, wherein said amount is effective to immunize asusceptible mammal against Ross River Virus infection and induces aprotective immune response in the host.

In accomplishing these and other objects of the present invention, thereare provided in one aspect of the invention a method for the productionof purified Ross River Virus antigen. This method comprises the steps ofinfecting a cell culture of cells with Ross River Virus, incubating saidcell culture to propagate said virus, harvesting the virus produced, andfiltering the harvested virus.

The cells used for infection of the virus can be any cell which issusceptible to RRV. According to one aspect of the invention the cellsare a continuous cell line of monkey kidney cells, such as VERO cells orCV-1 cells. VERO cells are available from the American Tissue CellCulture, deposited as ATCC CCL81.

Some substances like serum and serum-derived additives that cancontaminate the final harvested viral antigen may either be derived fromthe cell culture medium or the cells. Conventional cell culture mediacomprise serum or protein additives such as albumin, transferrin orinsulin, and other proteins or polypeptides derived from the serum oradded to the cell culture medium during cell growth.

According to one embodiment of the invention the cells are grown in aserum free medium. The medium can be a minimal medium, such as DMEM orDMEM HAM's F12 and other minimal media known in the art, such asdescribed in Kistner et al. (1998. Vaccine 16:960-968), which do notcomprise any serum additives.

In a preferred embodiment of the invention the cells are grown in aserum and protein free medium prior to infection as described in WO96/15213, WO 00/0300 or WO 01/123527, whereby said minimal medium can besupplemented with extracts of yeast or soy peptone.

The cells can be bound to microcarrier during cell culture growth. Themicrocarrier can be a microcarrier selected from the group ofmicrocarriers based on dextran, collagen, plastic, gelatine andcellulose and others as described in Butler (1988. In: Spier &Griffiths, Animal cell Biotechnology 3:283-303). Therefore, according toone embodiment of the invention the serum free or serum and protein freecells are cultivated and infected on microcarriers. Preferably, themicrocarrier are selected from the group of smooth surface such asCYTODEX I®, CYTODEX II® and CYTODEX III®, CYTOPORE® or CYTOLINE® (allPharmacia).

The cells bound to microcarrier are infected with RRV at a multiplicityof infection (m.o.i.) between about 0.001 and about 5.

Various conventional methods, such as chromatography, gradientcentrifugation etc, are known in the art to remove contaminatingproteins from the biological that is desired to be isolated andpurified. Efficient purification methods often comprise several stepsand combinations of filtration, ion exchange chromatography and gradientcentrifugation. However, the various methods may reduce the virus titerand antigen yield during each purification step.

Filtration is used in the art to purify biological material, e.g. toremove contaminating agents, or during preparation of virus-free bloodproducts to remove potential contaminating viruses, whereby viruses,particular enveloped-viruses, remain in the retenate, and the virustiter in the filtrate is reduced.

It has been surprisingly found by the present invention that byfiltering the cell culture supernatant derived from cells infected withenveloped viruses (e.g., the Ross River Virus), the enveloped viruspasses the filter system without reduction of virus titer, whilecellular contaminants, like proteins and nucleic acid are efficientlyremoved. The method of the invention provides purification of a hightiter virus preparation by filtration, wherein this method is easilyapplicable for large-scale purification and efficiently removes most ofthe protein derived from the host cells as well as of cellular nucleicacid. The method of the invention therefore provides a process ofpurifying virus antigen by filtering without remarkable loss of virustiter and virus antigen.

The methods of the invention can be used to purify any envelopedviruses. Exemplary viruses include, Alphaviruses (e.g., Ross RiverVirus, Eastern equine encephalitis Virus, Venezuelan equineencephalitis, Western equine encephalitis, Sindbis Virus, SemilikiForest Virus); Flaviviruses (e.g., St. Louis encephalitis Virus,Japanese encephalitis Virus, Dengue Virus, Yellow fever Virus,Tick-borne encephalitis Virus); Orthomyxoviruses (e.g., InfluenzaVirus); and Paramyxoviruses (e.g., New Castle Disease Virus).

According to one aspect of the methods of the invention, the filteringis performed on a filter having a pore size between about 0.3 and about1.5 μm, which is preferably a filter based on a positively chargedmatrix. According to another aspect of the invention the filtering isperformed on a filter having a pore size between about 0.1 and about 0.5μm, which is preferably based on a hydrophilic matrix.

According to a preferred embodiment the filtering is performed by acombination of at least two filters, a first having a pore size ofbetween about 0.3 and about 1.5 μm, and a second filter having a poresize of between about 0.1 and about 0.5 μm. The combination of thefiltering steps can be performed with a first filtering step on a filterhaving a pore size of between about 0.3 and about 1.5 μm and the secondfiltering on a second filter having a pore size of between about 0.1 andabout 0.5 μm. The filtering by a combination of filters can be performedeither sequentially or in separate steps. The filter can be a filtersuch as a positively charged depth filter having a pore size of about0.3 to about 1.5 μm and a hydrophilic filter having a pore size of about0.22 μm. Any filtration system known in the art can be used as well. Byfiltering during virus/virus antigen purification, substantially allcellular protein contamination is removed. The cellular contaminatingnucleic acid is also efficiently removed by a factor of at least 35, andan intermediate pure preparation having a purity of at least about 97%compared to the starting virus harvest is obtained by this purificationstep.

The filter used can be based on a cellulose fiber matrix, hydrophilicfilters, such as based on polyvinylidene fluoride membrane, or filtersbased on polypropylene membrane. Such filters are commerciallyavailable, e.g. ZETAPLUS® (CUNO), DURAPORE®, MILLIPAK® or MILLIDISK™(Millipore) or filters from Pall.

The method of the invention as described above therefore provides for apurified Ross River Virus antigen which is substantially free ofcontaminating proteins and nucleic acid from the cells or the cellculture. The preparation has a purity of at least about 97% compared tothe starting material.

Another aspect of the present invention relates to a method forproduction of a purified Ross River Virus comprising the steps ofinfecting a cell culture of cells with Ross River Virus, incubating saidcell culture to propagate said virus, harvesting the virus produced,filtering the harvested virus, treating the virus filtered with anucleic acid degrading agent and purifying the virus.

The virus preparation obtained after filtering is treated with a nucleicacid degrading agent to destroy the structural integrity of the nucleicacid by breaking down residual nucleic acid that have not been removedby filtering. The degradation of the nucleic acid ensures that highermolecular weight nucleic acids are broken down to molecules of lowermolecular weight, which are then removed during the final purificationstep.

The nucleic acid degrading agent according to the invention can be anenzyme which degrades nucleic acid, preferably a nucleic aciddegradation enzyme, such as a nuclease, having DNase and RNase activity,or an endonuclease, such as from Serratia marcescens, commerciallyavailable as BENZONASE® endonucelase (Benzon Pharma A/S). Mostpreferably the nucleic acid degradation agent is BENZONASE®endonucelase.

The virus treated with a nucleic acid degradation agent can be furthertreated with an inactivating agent. The inactivating agent can be anyagent with inactivating activity known in the art, such as e.g.formalin, BEI, laser light, UV light, chemical treatment such asmethylene blue, psoralen, carboxyfullerene (C60), or a combination ofany thereof as described in the prior art (Rowland et al. (1972). Arch.Ges. Virusforsch. 39:274-283; Mowat et al. (1973). Arch. Ges.Virusforsch. 41:365-370, Rweyemamu et al. (1989). Rev. Sci. Tech. Off.Int. Epiz. 8:747-767 and WO 01/46390). Other known methods in the artfor inactivating viruses can be used as well.

The treatment of the virus with the nucleic acid degrading andinactivating agent can be performed by a sequential treatment or in acombined/simultaneous manner, whereby the purified virus antigen istreated with a combination of BENZONASE endonuclease as nucleic aciddegrading agent and formalin or UV or BEI or a combination offormalin/BEI or formalin/UV as inactivation agent. The nucleic aciddegrading agent is preferably added to the virus preparation prior theaddition of the inactivation agent and during the course of theinactivation process, the nucleic acid degrading agent can be furtheradded subsequently, if necessary.

According to another embodiment of the method of the invention theinactivated virus is further purified. The method therefore comprisesafter the step of nucleic acid degradation/virus inactivation treatmenta further step to remove the nucleic acid degrading agent and theinactivating agent from the virus preparation. This can be performed byany method known in the art, such as chromatography, gel filtration orgradient centrifugation. According to one embodiment of the inventiongradient purification, such as sucrose gradient centrifugation ispreferred. This final purification step also removes the break-downproducts of the nucleic acid treatment with nucleic acid degrading agentand removes the residual nucleic acid and proteins that have not beenremoved by the filtering.

The preparation obtained with this method comprising Ross River virusantigen, wherein said preparation is substantially free of contaminatingproteins derived from the cells or the cell culture and has less thanabout 50 pg cellular nucleic acid/μg virus antigen, preferably less thanabout 20 pg and most preferred less than about 10 pg cellular nucleicacid/μg virus antigen. Purified Ross River Virus antigen obtained isfree of contaminating proteins and nucleic acid, suitable for humanclinical use and is stable.

Another aspect of the invention provides a method for production of avaccine comprising purified, inactivated Ross River Virus antigencomprising the steps of infecting a cell culture of cells with RossRiver Virus, incubating said cell culture to propagate said virus,harvesting the virus produced, filtering the harvested virus, treatingthe filtered virus preparation with a nucleic acid degrading agent andvirus inactivating agent, purifying the virus and formulating thepurified and inactivated virus in a vaccine composition. According toone embodiment of the invention the method provides for a vaccinecomprising purified

Ross River Virus antigen and being substantially free of contaminatingproteins and nucleic acids.

According to another aspect, the invention provides for a preparationcomprising purified Ross River Virus antigen being substantially free ofcontamination from the cells or cell culture.

According to another aspect, the invention provides for a preparationcomprising purified Ross River Virus antigen being substantially free ofcontaminating proteins from the cells or cell culture. The preparationhas less than about 50 pg cellular nucleic acid/μg virus antigen,preferably less than about 10 pg nucleic acid/μg virus antigen.According to one embodiment of the invention the preparation comprises aphysiologically acceptable carrier, such as a phosphate or Tris-basedbuffer.

In accordance with another aspect of the invention, there is provided avaccine against Ross River Virus infection comprising a host protectiveamount of Ross River Virus antigen in an amount of between 0.1 to 50 μgantigen/dose, preferably between 0.3 and 30 μg antigen/dose. The antigencan be a whole virus or a fragment of the virus, such as a peptide orpolypeptide, having an immunogenic epitope to induce protectiveantibodies against RRV infection. In a preferred embodiment of theinvention the vaccine comprises as RRV antigen as whole inactivatedvirus.

The vaccine of the present invention comprising highly purified RRV canfurther comprise an adjuvant. It has been found that the presence of anadjuvant increases the immunogenicity of the purified antigen of theinvention by inducing higher titers of neutralizing, protectiveantibodies when compared to a vaccine composition which does notcomprise an adjuvant. The beneficial effect of the present vaccine maybe due the higher purity of the RRV antigen used in the vaccine. Theadjuvant may be aluminum or a salt thereof, mineral oils, Freundadjuvant, vegetable oils, water-in-oil emulsion, mineral salts,immunomodulator, immunopotentiator or other well known adjuvant known inthe art at a desired concentration to increase the immune response e.g.to stimulate the production of neutralizing antibodies. The amount ofadjuvant is well within the level of skill.

In a preferred embodiment of the invention, the adjuvant is aluminum ora salt thereof, such as aluminum hydroxide or aluminum phosphate. Thealuminum concentration is preferably between 0.001% and 1% (w/v) perdose. The vaccine may be formulated in the most varying manner.

The vaccine may be administered in any known manner e.g. subcutaneously,intramuscularly or intraperitoneally. According to a preferredembodiment the vaccine is administered intramuscularly in a vaccinationscheme in humans of 0, 1, 6 or 12 months or in a rapid immunizationscheme for travelers of about 0, 14 and 28 days with a higher antigendose per immunization and booster.

Another aspect of the invention provides for a method of immunizing amammal against Ross River Virus infection comprising the steps ofproviding a vaccine comprising a host protective amount of purified RossRiver Virus antigen and having an amount of cellular DNA of less than 10pg/μg antigen, and wherein said vaccine is substantially free of anycontaminating protein from the cells or the cell culture andadministering said vaccine to a mammal.

According to a further aspect of the invention there is provided amethod for the preparation of an immune globulin preparation comprisingantibodies specific against Ross River Virus. The preparation comprisingthe immune globulins is obtained by immunizing a mammal with a RossRiver Virus Vaccine as described above and isolating from the serum ofthe immunized mammal the immune globulin fraction comprising the RRVspecific antibodies. The immune globulin fraction can be isolated byconventional method known in the art, such a Cohn fractionation and ionexchange chromatography.

These and other aspects of the invention disclosed herein will becomeapparent to the skilled artisan in view of the disclosure containedherein.

Example 1 Production of Purified, Inactivated Ross River Virus Antigen

a) Production of Ross River Virus on Serum and Protein Free VERO CellCulture.

VERO cells (African Green Monkey, Ceropthecus aethiops, kidney) are usedas a production cell line. The cells are obtained from the American TypeCell Culture Collection, Rockville, Md. at a passage number 124 underthe designation ATCC CCL81. The cells are adapted to grow in serum orprotein free medium as described in Kistner et al., 1998 (supra) WO96/15231. For growth in serum free medium a basal DMEM HAM's F12 mediumsupplemented with inorganic salts, amino acids, sodium bicarbonate andyeast extract is used. The working cell bank is prepared without the useof any animal derived medium components. Cells of the working cell bankare expanded in T-flasks and roller bottles with a split ratio of 1:6.Further propagation of the cells is performed in a stirred tankbioreactor using CYTODEX® microcarrier as attachment substrate. Thecells are grown at 37° C. The culture conditions of oxygen saturation20%+/−10% and pH7.25+/−0.35 are kept constant during virus propagationprocess. A serum free cell culture system of VERO cells as described byKistner et al., (Vaccine 16:960-968 (1998)) is infected with Ross RiverVirus at a multiplicity of infection (m.o.i.) of 0.001. After anincubation time of 3 days at 37° C. the fermenter is harvested and virusis recovered from the cell culture supernatant. The harvested virus gavea titer of 8.0 TCID₅₀/ml after removal of microcarriers and cell debrisby centrifugation (about 9000 g).

b) Purification of RRV

The harvested virus is purified by filtering on a combination of a 1.2μm filter (ZETAPLUS®, CUNO) and a 0.22 μm filter (DURAPORE®, Millipore).The efficacy of the filtering to remove soluble proteins derived fromthe cell culture, in particular from VERO cells, is determined bywestern blot analysis with VERO cell protein specific antibodies (FIG.1A). FIG. 1 impressively shows that the filtering removes substantiallyall VERO cell derived proteins from the virus preparation.

Determination of virus titer of the virus harvest before filtering is8.0 TCID₅₀/ml and after the filtering 7.4 TCID₅₀/ml (Table 1) revealedthat during the filtering step substantially no virus antigen is lost.Detection of RRV antigen by western blot analysis with RRV specificantibodies of the same samples as used for detection of VERO cellprotein (FIG. 1B) showed that similar amounts of virus antigen is foundin all samples tested during the various purification steps, indicatingno loss of virus antigen during purification.

The amount of VERO cell DNA is determined by PCR as described in U.S.Pat. No. 5,858,658 is determined after each purification step andsummarized in Table 1. The results show that during the filtering stepthe contaminating cellular nucleic acid is removed by a factor of atleast 35.

After the filtering, BENZONASE endonuclease (2000 U/I) is added to thevirus harvest to digest residual VERO cell and viral nucleic acid.During the following treatment with BENZONASE endonuclease the residualcontaminants are removed by a factor of at least 2.

TABLE 1 Determination of virus titer and contaminating nucleic acidduring Ross River Virus antigen purification process Total Protein VeroVirus titer Amount cell DNA DNA/Protein Purification Step (TCID₅₀/ml)(μg/ml) (pg/ml) (pg/μg) Harvest 8.0 86 54 × 10⁴ 6.300 Separator 7.6 8134 × 10⁴ 4.200 Filtration 7.2 80 14 × 10³ 175 1.2 mm/0.2 mm BENZONASE7.4 85  7 × 10³ 82 endonuclease Sucrose gradient inactivated 180  1 ×10³ 5.5

After 1 h incubation at 37° C. with BENZONASE endonuclease, formalin, asa virus inactivation agent with an end concentration of 0.1% (w/v), isadded for total inactivation time of 120 h at 37° C. Twenty-four hoursafter the addition of formalin, BENZONASE endonuclease (1000 U/I) isadded again. Filtration steps on 0.22 μm sterile filter (Millipore) areperformed at 0, 2, 6, 24, 48, 72, 96, 120, 144, 168 and 192 hours andsamples for virus titration and safety experiments are drawn inparallel.

The virus titration is carried out on VERO cells by determination of thetissue culture infectious dose 50 (TCID₅₀). Safety tests fordemonstration of complete virus inactivation are carried out on C6-36cells because they are about 10-100 times more susceptible for infectionsusceptible for infection with RRV than VERO cells.

The preparation comprising the inactivated virus is subjected to largescale flow zonal centrifugation over a 0-50% sucrose gradient to removeresidual formalin, BENZONASE endonuclease and nucleic acid break downproducts derived from the BENZONASE endonuclease treatment. The sucrosegradient purification of inactivated virus resulted in one peak atsucrose concentration between 40% and 42%, demonstrating a pure andhomogeneous antigen preparation. The fractions containing the virus arepooled and subjected to a 0.2 μm sterile filtration step.

The overall reduction during the purification process aftersucrose-gradient therefore allowed reduction of contaminating nucleicacid by at least 4 log steps. The final purified RRV preparationcontained about 5.5 pg cellular DNA/μg virus antigen. The isolatedpreparation of RRV antigen has a purity of at least 98% with regard tocellular contaminants. The final purity of this preparation with regardto nucleic acid is also given in Table 1.

Example 2 Characterization of RRV

The inactivated RRV preparation of Example 1 is adjusted to a proteinconcentration of 10 μg/ml. The endotoxin content, determined byLAL-assay, is less than 1.50 EU/ml. The pyrogenicity test is performedaccording to European Pharmacopoeia 2001, 2.6.8. A temperature increaseof greater than 2.65° C. is considered as pyrogen, and smaller than1.15° C. as pyrogen free. The amount of VERO cell DNA is determined byPCR as described in U.S. Pat. No. 5,858,658. The amount of residual VEROcell DNA in the preparation is far below the limit required by the WHOfor biologicals produced in continuous cell lines (CCLs), i.e. 10 ng perdose (1998, WHO, Technical Report Series No. 878). The results of thecharacterization of purity of the inactivated RRV preparation is shownin Table 2.

TABLE 2 Analysis of RRV Vaccine Test Result Dose/Antigen 10 μg LAL <1.50EU/ml Pyrogenicity 0.50° C. (pyrogen free) VERO cell DNA 50 pg

Example 3 Immunogenicity of the RRV Vaccine

Determination of the Effective Dose (ED₅₀) of antigen is performed byadjusting the RRV antigen concentration to 10 μg/dose, without and withAl(OH)₃ as an adjuvant at concentration of 0.05%, 0.1% and 0.2% (w/v).The candidate vaccine preparation is then diluted in 4 fold steps. Eachdilution is injected into a group of 10 CD1 mice. After 4 weeks, themice are boostered with the respective amount of antigen. Blood samplesare drawn at 4 weeks, before the booster, and at 6 weeks after thebooster. The sera of the samples are analyzed by an RRV-antibody ELISAand the ED₅₀ is calculated. Table 3 shows the ED₅₀ of the vaccine withand without adjuvant. To induce an immune response with similar antibodytiters, in the vaccine comprising an adjuvant only ⅕ to 1/20 of theamount of antigen of the vaccine with adjuvant is needed, depending onthe adjuvant concentration used. Increasing the adjuvant concentrationin the final preparation allows reduction of virus antigen amount in thevaccine. This is in contrast to prior art results of Yu et al. andAaskov et al. (supra), which had showed negative influence of adjuvanton protective antibody induction.

TABLE 3 Effective dose (ED₅₀) and protective dose (PD₅₀) of RRV vaccinein mice ED₅₀ ED₅₀ PD₅₀ Antigen (ng) Antigen (ng) Antigen (ng) Adjuvant 4weeks Booster 6 weeks 6 weeks Al(OH)₃ 413 150 1250 — 83 2 20 0.05%  74 920 0.1% 20 7 20 0.2%

The sera of the mice used for determination of ED₅₀ are also analyzed interms of their neutralizing activity. The neutralization assay isperformed by diluting infectious virus in 10 fold steps and incubatedeither with buffer or with heat-inactivated 1:10 dilutions of mouseserum for 1 hour at RT. The virus dilutions are subjected to a plaqueassay on VERO cells to determine the virus titer. The neutralizationindex or ratio of virus titer in buffer as control versus virus titerincubated with mouse serum is calculated. The results show that lessantigen in the vaccine composition comprising adjuvant is needed toinduce higher Neutralization Titer (NT) compared to the vaccinecontaining adjuvant.

Determination of Protective Dose (PD₅₀) is performed in that half of theimmunized mice of each group used in the previous experiment which arechallenged at week 6 and 2 weeks after booster with 10⁶ TCID₅₀ ofinfectious RRV. The results of the experiment show that 50% of the micebeing infected with infectious RRV have not developed a viraemia at therespective antigen dose. These results are given in Table 3.

These results show that the presence of an adjuvant in the vaccine didnot effect the induction of a protective immune response. Even more, thevaccine preparation of the present invention allowed a drastic reductionof the antigen content in the vaccine dose of up to 1/20 in the presenceof an adjuvant compared to the vaccine without adjuvant. Therefore, thevaccine preparation of the present invention differed in regard topurity and capability to induce protective immune response in thepresence of adjuvant from those known in the prior art.

Accordingly, the amount of protective dose for larger mammal will be inthe range between 0.1 and 50 ng antigen/protective dose depending on theaverage body weight of the mammal.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference for all purposes.

What is claimed is:
 1. A preparation comprising purified Ross RiverVirus antigen being free of contaminating protein from the cells or thecell culture and has less than 10 pg cellular nucleic acid/μg virusantigen.
 2. The preparation according to claim 1, further comprising aphysiologically acceptable carrier.
 3. The preparation according toclaim 1, further comprising an adjuvant.
 4. A vaccine against Ross RiverVirus infection comprising a host protective amount of a purified RossRiver Virus antigen, wherein said vaccine is substantially free of anycontaminating protein from the cells or the cell culture and has anamount of cellular nucleic acid per vaccine dose of less than 10 pg/μgantigen.
 5. The vaccine according to claim 4, wherein said hostprotective amount of Ross River Virus antigen is between about 0.1 andabout 50 μg/dose.
 6. The vaccine according to claim 4, furthercomprising an adjuvant.
 7. A method of immunizing a mammal against RossRiver Virus infection comprising the steps of providing a vaccinecomprising a host protective amount of Ross River Virus antigen, whereinsaid vaccine is substantially free of any contaminating protein from thecells or the cell culture and has an amount of cellular DNA of less thanabout 10 pg/μg antigen, and administering said vaccine to a mammal.
 8. Amethod for the preparation of an immune globulin preparation specificagainst Ross River Virus comprising the steps of (i) immunizing a mammalwith a vaccine according to claim 4 and (ii) isolating from the serum ofthe immunized mammal the immune globulin fraction comprising the RRVspecific antibodies.